Corticotropin-releasing hormone (CRH, also called CRF or corticoliberin) was first characterized as a 41-residue peptide isolated from ovine hypothalami by Vale et al. (1981). Subsequently, the sequence of human-CRH was deduced from cDNA studies and shown to be identical to rat-CRH, and then caprine, bovine, porcine, and white sucker fish CRH were characterized. The CRH of hoofed animals show considerable differences from man, but the pig and fish sequences differ from the human/rat sequence by only 2 out of 41 residues.
For some mysterious reason, peptides with homologous structures to mammalian CRH are found in cells of certain frog skins and in the urophysis of fish. In fact, the structure of sauvagine, the 40-amino acid peptide isolated from the skins of Phyllomedusa frogs, was reported several years before Vale's description of ovine-CRH. The structure of sucker fish urotensin I was reported just months after the description of ovine-CRH and resulted from an independent line of inquiry by Lederis's group in Canada. Although sauvagine and urotensin I release adrenocorticotropin (ACTH) from the pituitary, the functions of these peptides in the tree-frog (Phyllomedusa species that live in arid regions of South America) and in the sucker fish remain unknown.
TABLE 1 Peptides of the Corticotropin-Releasing Hormone Superfamily SEQ. ID NO. PEPTIDE SPECIES SEQUENCE.sup.a,b 1 CRH Human/ SEEPPISLDL TFHLLREVLE MARAEQLAQQ AHSNRKLMEIJ rat 2 CRH Pig SEEPPISLDL TFHLLREVLE MARAEQLAQQ AHSNRKLMENF 3 CRH Sucker SEEPPISLDL TFHLLREVLE MARAEQLAQQ AHSNRKMMEIF fish 4 CRH Sheep/ SQEPPISLDL TFHLLREVLE MTKADQLAQQ AHSNRKLLDIA goat 5 CRH Cow SQEPPJSLDL TFHLLREVLE MTKADQLAQQ AHNNRKLLDIA 6 Urotensin I Sucker NDDPPISIDL TFHLLRNMIE MARIENEREQ AGLNRKYLDEV fish 7 Urotensin I Carp NDDPPISIDL TFHLLRNMJE MARIENEREQ AGLNRKYLDEV 8 Urotensin I Maggy SEEPPMSJDL TFHMLRNMIH RAKMEGEREQ ALJNRNLLDEV sole 9 Urotensin I European SEDPPMSIDL TFHMLRNMJH MAKMEGEREQ AQINRNLLDEV flounder 10 Urocortin Rat DDPPLSIDL TFHLLRTLLE LARTQSQRER AEQNRJIFDSV 11 Urocortin Human DNPSLSIDL TFHLLRTLLE LARTQSQRER AEQNRIJFDSV 12 Sauvagine Frog &gt;EGPPISJDL SLELLRKMIE IEKQEKEKQQ MNNRLLLDTI .sup.a The carboxyl termini of these peptides are amidated. .sup.b Single letter abbreviations for amino acids: S, T, P, A, G; Ser, Thr, Pro; Ala, Gly; M, L, I, V; Met, Leu, IIe, VaI; E, D, N, Q; Glu, Asp, Asn, Gln; R, K, H; Arg, Lys, His; F, Y, W, Phe, Tyr, Trp; &gt;E; pyroglutamyl
In humans CRH regulates, via release of proopiomelanocortin, ACTH secretion from the anterior pituitary and has several direct actions on central and peripheral tissues. CRH has also been found to have direct anti-inflammatory properties. More recently, evidence has been provided that mammalian skin cells both produce CRH and express functional CRH receptors (Slominski et al., FEBS Lett., 374, pp. 113-116, 1995; Slominski et al., J. Clin. Endocrinol. Metab., 83, pp. 1020-1024, 1998; Slominski et al., Hum. Pathol., 30, pp. 208-215, 1999), although it was not known whether locally produced CRH had an additional role in the physiology of the skin, other than as a vasodilator and inhibitor of thermal injury-induced edema.
Some therapeutic methods and uses for CRH are described by inventors Wei and co-workers in U.S. Pat. No. 4,801,612, issued Jan. 31, 1989, titled "Method of Inhibiting Inflammatory Response," and U.S. Pat. No. 5,137,871, issued Apr. 26, 1994, titled "Treatment to Reduce Edema for Brain and Musculature Injury." These patents describe the use of CRH to decrease the leakage of blood components into tissues produced by various adverse medical conditions, and thus to treat a patient for injury to or disease ofthe brain, central nervous system or musculature in which edema is a factor.
U.S. Pat. No. 5,869,450, issued Feb. 9, 1999, inventors Wei et al., describes CRH analogs in which the fifth amino acid from the N-terminus is D-Pro or in the case of urocortin or sauvagine where the fourth amino acid from the N-terminus is D-Pro or D-Ser. These analogs have an anti-inflammatory activity and a disassociated ACTH response.
Cyclic CRH agonists have recently been described by Rivier et al. (U.S. Pat. Nos. 5,844,074 and 5,824,771). These CRH analogs, modified by cyclization of residues 30-33 of CRH via a glutamic acid-lysine bridge, are more potent than native CRH in the release of ACTH and have lower molecular weight than native CRH. The elimination of residues 1-3 or 1-11 at the N-terminus of CRH has been shown to not alter biological activities or ACTH-release potency. (Kornreich et al., J. Med. Chem., 35, pp. 1870-1876, 1992; Koerber et al., J. Med. Chem., 41(25), pp. 5002-5011, 1998.)
Recently, Tjuvajev et al. (Tjuvajev, J., Kolesnikov, Y., Joshi, R., Sherinski, J., Koutcher, L., Zhou, Y., Matei, C., Koutcher, J., Kreek, M. J., and Blasberg, R. Anti-neoplastic properties of human corticotropin releasing factor: involvement of the nitric oxide pathway. In Vivo., 12, pp.1-10, 1998. Department of Neurology, Memorial Sloan Kettering Cancer Center, New York, N.Y. 10021, USA) have introduced yet another novel mechanism for CRH in the form of anti-cancer action. Tjuvajev et al. (1998) reported a series of in vivo and in vitro studies that evaluated the anti-neoplastic potential of CRH in W256 rat mammary carcinoma. Using magnetic resonance imaging (MRI) and direct measurements of tumor and peritumoral brain water content they found that CRH treatment (100 micrograms/kg subcutaneously twice a day for 3 days) caused significant inhibition of growth of intracerebrally-injected W256 tumor cells. CRH also exhibited antiproliferative effects in in vitro cultures of W256 cells. The antiproliferative effects of CRH in W256 cells involve activation of nitric oxide synthase (NOS) and L-arginine-NO pathways. CRH activated the release of NO in W256 cells. The NO then became cytotoxic to the cancer cells.
Human trials of CRH for the treatment of peritumoral brain edema have been initiated and preliminary data indicated that CRH reduced brain edema associated with tumor metastases. However, the limiting factor on the use of CRH has been the known blood-pressure lowering property of CRH. CRH causes relaxation of smooth muscles surrounding blood vessels and causes vasodilation, resulting in a lowering of blood-pressure. The hypotension so produced is sufficiently dangerous to limit the dosages of CRH that can be administered to humans. If this dose-limiting toxicity is overcome by improved molecular design of CRH superfamily molecules, then it is conceivable that these analogs will have a higher therapeutic index and have utility via the anti-proliferative mechanism of action.